[17] analyses of Ras effector utilization in cellular transformation
195
[17] Genetic and Pharmacologic Dissection of Ras
Effector Utilization in Oncogenesis
By PAUL M. CAMPBELL, ANURAG SINGH, FALINA J. WILLIAMS,
KAREN FRANTZ, AYLIN S. ÜLKÜ, GRANT G. KELLEY, and CHANNING J. DER
Abstract
Ras proteins function as signaling nodes that are activated by diverse
extracellular stimuli. Equally complex for this family of molecular switches
is the multitude of downstream effectors and the pathways that they
traverse to translate extracellular signals into a spectrum of cellular consequences. To better understand the individual and collective roles of these
effector signaling networks, both genetic and pharmacological tools have
been developed. By either stimulating or ablating specific components in a
cascade downstream of Ras activation, one can gain insight into the specific
signaling underlying a particular Ras phenotype, for example, malignant
transformation. In this chapter, we describe the use of activating and dominant‐negative mutations, both artificial and naturally occurring, of Ras and
its effectors, as well as pharmacological inhibitors used to probe the effector pathways (Raf kinase, phosphoinositol 3‐kinase, Tiam1, phospholipase
C epsilon, and RalGEF) implicated in Ras‐mediated oncogenesis.
Introduction
Ras proteins (H‐, K‐ and N‐Ras) function as GDP/GTP‐regulated signaling nodes. These proteins are activated by extracellular stimuli capable
of triggering the signaling cascades emanating from a variety of cell surface
proteins, including receptor tyrosine kinases, integrins, and G protein–
coupled receptors (Malumbres and Barbacid, 2003). In addition, there is a
complex plethora of effector molecules that function downstream of Ras
(Feig and Buchsbaum, 2002; Malumbres and Barbacid, 2003; Repasky et al.,
2004). A Ras effector binds preferentially to the activated GTP‐bound form
of Ras and requires an intact core effector domain (Ras residues 32–40).
Most Ras effectors possess Ras‐binding domains (RBDs) or Ras association
(RA) domains. The main Ras effector classes that have been found to
contribute to Ras‐mediated transformation are the Raf serine/threonine
kinases, phosphatidylinositol 3‐kinases (PI3K), Ral guanine nucleotide
exchange factors (RalGEFs), Tiam1, and phospholipase C epsilon (Fig. 1).
Ras activation leads to many facets of the complex phenotype of the
cancer cell. Critical to the understanding of Ras signaling are the molecular
METHODS IN ENZYMOLOGY, VOL. 407
Copyright 2006, Elsevier Inc. All rights reserved.
0076-6879/06 $35.00
DOI: 10.1016/S0076-6879(05)07017-5
196
regulators and effectors of small GTPases: Ras family
[17]
FIG. 1. Effector signaling pathways that contribute to Ras‐mediated transformation.
and pharmacological tools that facilitate teasing out the contribution of
individual effector components. Our laboratory and others have used
several of these tools and methodologies to explain the role of specific
downstream effector signaling pathways in oncogenic Ras‐mediated
growth transformation, tumorigenesis, invasion, and metastasis with the
long‐term goal of identifying potential targets for therapeutic intervention.
To reveal the necessity of an effector pathway, various pharmacological
and genetic approaches (dominant negative mutants, short interfering
RNA [siRNA], genetically modified mice) can be used to selectively block
the activity of that specific pathway. To address whether an effector pathway alone is sufficient to mediate a specific aspect of Ras‐dependent
oncogenesis, Ras effector domain mutants, constitutively activated effectors, or effector substrates can be used. We have summarized some of the
reagents that we have applied or developed to address the role of particular effector function in Ras‐mediated morphological and growth transformation, and we cite examples from our analyses of rat ovarian surface
epithelial (ROSE) and fibroblast cells.
Reagents for Assessment of Effector Sufficiency
H‐Ras Effector Domain Mutants
Developed initially by White and colleagues, the use of H‐Ras effector
domain mutants that are differentially impaired in effector activation has
provided a powerful tool to determine the role of specific effectors in Ras
[17] analyses of Ras effector utilization in cellular transformation
197
function (Joneson et al., 1996; Khosravi‐Far et al., 1996; Rodriguez‐Viciana
et al., 1997; White et al., 1995) (Fig. 1, Table I). We have also generated
similar mutants of activated K‐Ras and N‐Ras, although their differential
activation of the Raf and PI3K effector pathways is not as distinct as has
been seen with the H‐Ras mutants (Vos et al., 2003; Wolfman et al., 2002).
Our pBabe‐puro (or pBabe‐hygro) retrovirus‐based mammalian expression vectors (Morgenstern and Land, 1990) were generated by the
following procedures. Polymerase chain reaction (PCR)–mediated DNA
amplification of cDNA sequences from pDCR‐H‐Ras(12V) expression
plasmids using a 50 primer containing a BamHI site and a 30 primer containing an EcoRI site generated a 590‐base pair fragment (McFall et al., 2001).
The products were digested with BamHI and EcoRI and ligated into the
BamHI and EcoRI sites of pBabe‐puro. Plasmids that express H‐Ras(12V)
effector domain mutants were constructed in a similar fashion on the
pBabe‐puro backbone. These include H‐Ras(12V/35S), H‐Ras(12V/37G),
and H‐Ras(12V/40C), which are altered in their activation of Raf kinase,
RalGEF, and PI3K effector signaling (McFall et al., 2001). The H‐Ras
(12V/35S) mutant retains the ability to activate Raf but not PI3K or
RalGEF. The H‐Ras(12V/37G) mutant no longer activates Raf or PI3K
but can activate RalGEF. The H‐Ras(12V/40C) mutant can activate PI3K
but not Raf or RalGDS.
A caution about the use of these effector domain mutants is that their
selective activation of a subset of effectors may vary when expressed in
different cell types. Hence, it should be validated that they retain selective
activation of ERK, AKT, and RalA‐GTP in the cell type used. A second
caveat is that these mutants do retain binding to other Ras effectors. For
example, H‐Ras(12V/37G) still binds and activates other effectors such as
PLC" (Kelley et al., 2001) and Rin1 (Wang et al., 2002). Hence, an activity
associated with the expression of this mutant may not necessarily be
ascribed to RalGEF activation alone or at all.
Constitutively Activated Mutant Effectors
A second approach that can complement the use of Ras effector domain mutants is the use of constitutively activated Ras effectors. The
activated effectors are described in Table I. A potential advantage of using
activated effectors is that in principle no other effector pathway is activated
concurrently as with the Ras effector domain mutants. However, this does
not exclude the possibility of cross‐talk and activation of components
associated with other effector pathways. A potential disadvantage is that
they may not fully mimic Ras activation of that effector class. For example,
N‐terminally deleted (Raf‐22W) or plasma membrane–targeted c‐Raf‐1
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regulators and effectors of small GTPases: Ras family
[17]
TABLE I
REAGENTS FOR ACTIVATION OF RAS EFFECTOR PATHWAYS
Effector/substrate
Description
Reference
Raf‐MEK‐ERK pathway
H‐Ras(G12V/35S)
Oncogenic form of H‐Ras that binds to and
activates c‐Raf‐1 kinase but not PI3K or
RalGDS
Raf‐22W
N‐terminal 305 amino acid truncated
version of human Raf‐1 that lacks the
RBD cysteine‐rich domain Ras‐binding
sequences. Encodes an approximately
39‐kDa protein
Human c‐Raf‐1 chimeric protein
terminating with C‐terminal 18 amino
acid plasma membrane targeting
sequence of K‐Ras4B
Human B‐Raf with the V600E (formerly
V599E) missense mutation seen in most
mutated B‐Raf alleles found in human
cancers
Human c‐Raf‐1 with a missense mutation
at tyrosine 340 to mimic constitutive
phosphorylation by Src family kinase
S218E and S222D missense mutation
of Raf phosphorylation sites to mimic
persistent phosphorylation; also
N‐terminal deletion of amino
acids 31–52
A hyperactive allele of ERK2, analogous
to the Drosophila sevenmaker
gain‐of‐function mutation, has significantly
reduced sensitivity to MAPK phosphatases
but does not possess significantly enhanced
intrinsic catalytic activity
Chimeric fusion protein of ERK2 and
MEK1 with a mutated nuclear export
sequence; partially activated
Raf‐CAAX
B‐Raf(V600E)
Raf(Y340D)
MEK2 MEKED
ERK2 (D319N)
ERK2‐MEK1‐LA
(Khosravi‐Far
et al., 1996;
Rodriguez‐Viciana
et al., 1997;
White et al., 1995)
(Stanton et al., 1989)
(Leevers et al., 1994;
Stokoe et al., 1994)
(Davies et al., 2002)
(Fabian et al., 1993)
(Mansour et al., 1994)
(Bott et al., 1994;
Chu et al., 1996)
(Bott et al., 1994)
PI3K‐AKT pathway
H‐Ras(G12V/40C)
Oncogenic form of H‐Ras that binds to and
activates PI3K but not c‐Raf‐1 kinase
or RalGDS
p110‐CAAX
Bovine p110 chimeric protein terminating
with C‐terminal prenylation signal
sequence of K‐Ras4B
(Khosravi‐Far
et al., 1996;
Rodriguez‐Viciana
et al., 1997;
White et al., 1995)
(Wennstrom and
Downward, 1999)
[17] analyses of Ras effector utilization in cellular transformation
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TABLE I (continued)
Effector/substrate
p110(K227E)
Description
Reference
Point mutation in Ras‐binding domain,
results in constitutively activated protein
Membrane‐targeted, constitutively activated
AKT1 containing the c‐Src N‐terminal
myristoylation signal sequence
(MGSSKSKPK)
(Rodriguez‐Viciana
et al., 1996)
(Eves et al., 1998;
Kohn et al., 1996)
H‐Ras(G12V/37G)
Oncogenic form of H‐Ras that binds to
and activates RalGDSs but not PI3K
or c‐Raf‐1 kinase
Rlf‐CAAX
Mouse Rlf/RGL2 chimeric protein
terminating with C‐terminal 18 amino
acids plasma membrane targeting
sequence of K‐Ras4B; Rlf lacks the
C‐terminal 247 residues that
contain the Ras association domain.
Human GTPase–deficient mutant
(Khosravi‐Far
et al., 1996;
Rodriguez‐Viciana
et al., 1997;
White et al., 1995)
(Wolthuis et al., 1997)
(Lim et al., 2005)
Human GTPase–deficient mutant
(Lim et al., 2005)
Human fast cycling mutant
(Lim et al., 2005)
Human GTPase–deficient mutant
Human GTPase–deficient mutant
Human fast cycling mutant
(Lim et al., 2005)
(Lim et al., 2005)
(Lim et al., 2005)
N‐terminally truncated, constitutively
activated
Human GTPase–deficient, constitutively
activated
Human GTPase–deficient, constitutively
activated
(Michiels et al., 1997)
Myr‐AKT
RalGEF‐Ral pathway
RalA(G23V or
G26V)a
RalA(Q72L or
Q75L)a
RalA(F39L or
F42L)a
RalB(G23V)
RalB(Q72L)
RalB(F39L)
Tiam1‐Rac pathway
Tiam1 C1199
Rac1(G12V)
Rac1(Q61L)
a
(Khosravi‐Far
et al., 1995)
(Khosravi‐Far
et al., 1995)
Two human RalA sequences have been identified, with one containing three additional
N‐terminal amino acids (Chardin and Tavitian, 1989; Polakis et al., 1989).
(Raf‐CAAX) are laboratory‐generated variants that may not fully recapitulate the activity caused by Ras activation of endogenous Raf, which
may consist of c‐Raf‐1 and additionally the A‐Raf and B‐Raf isoforms.
Although highly related in sequence, regulation, and function, the Raf
isoforms are nevertheless functionally distinct (Wellbrock et al., 2004a).
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regulators and effectors of small GTPases: Ras family
[17]
Similarly, because there are four RalGEFs (RalGDS, RGL, RGL2/Rlf,
and RGL3), ectopic expression of one RalGEF (e.g., Rlf‐CAAX) may not
fully mimic Ras activation of multiple, endogenous RalGEFs.
Because Ras activation of effector function is mediated, in part, by promoting the translocation of effectors from the cytosol to the plasma membrane (Hancock, 2003), a general approach to generate constitutively
activated effectors is the addition of a Ras C‐terminal (e.g., SKDGKKKKKKSKTKCVIM) plasma membrane targeting sequence (Cox and
Der, 2002) to generate Raf‐CAAX, p110‐CAAX, and Rlf‐CAAX. CAAX
refers to the C‐terminal prenylation signal sequence (where C ¼ cysteine,
A ¼ aliphatic amino acid, and X ¼ terminal amino acid) that together
with upstream polylysine residues constitute the two elements necessary
and sufficient for K‐Ras4B plasma membrane targeting. In this manner,
constitutively membrane‐targeted effector molecules can be created such that
individual effector pathways are stimulated without activation of Ras itself.
To generate a plasma membrane–targeted human c‐Raf‐1 expression
vector, a cDNA sequence encoding a plasma membrane–targeted version
of human c‐Raf‐1 was subcloned into the unique BamHI site of pBabe‐
puro (McFall et al., 2001). To stimulate the PI3K‐AKT serine/threonine
kinase pathway, a Myc epitope‐tagged version of the bovine p110 subunit
of PI3K with a K‐Ras4B C‐terminal targeting sequence (designated p110‐
CAAX) (Rodriguez‐Viciana et al., 1994) was subcloned into the BamHI
site of pBabe‐puro (McFall et al., 2001) or pBabe‐hygro (Williams and Der,
unpublished). For activation of the RalGEF‐Ral pathway, a hemagglutinin
(HA) epitope–tagged, plasma membrane–targeted form of mouse RGL2/
Rlf (designated Rlf‐CAAX) (Wolthuis et al., 1997) was subcloned into
EcoRI site of pBabe‐puro (McFall et al., 2001) or pBabe‐hygro (Williams
and Der, unpublished). When stably expressed in a variety of cell types, it
causes increased steady‐state levels of RalA‐GTP. The complete cDNA
and protein sequences for these activated effectors can be found at http://
cancer.med.unc.edu/derlab/methods.html.
In addition to membrane‐targeted c‐Raf‐1, we have also used several
other activated Raf variants. Raf‐22W is a truncated form of c‐Raf‐1
that lacks the N‐terminal 305 amino acids that inhibit the kinase domain
(Stanton et al., 1989). To create this reagent, a 1.98 kb EcoRI‐fragment
containing a 981 bp noncoding 30 region was cloned into the EcoRI site of
pBabe‐puro (McFall et al., 2001). Another weakly activated variant of
c‐Raf‐1 contains a missense mutation that mimics constitutive phosphorylation of tyrosine 340, a residue normally phosphorylated at the plasma
membrane by Src family kinases (Fabian et al., 1993). Finally, the recent
identification of mutationally activated B‐Raf in human cancers (Davies
[17] analyses of Ras effector utilization in cellular transformation
201
et al., 2002) provides a fourth transforming Raf variant. This construct was
generated by site‐directed mutagenesis (QuikChange, Stratagene, La Jolla,
CA) of the wild‐type cDNA (a gift of P. J. Stork, Oregon Health Sciences
University) in the pcDNA3 plasmid (Invitrogen, Carlsbad, CA) to convert
valine 600 to glutamic acid (V600E: formerly called V599E [Kumar et al.,
2004]), then subcloned into pBabe‐puro. Although no one activated variant
of Raf may accurately mimic the precise consequences of Ras activation of
endogenous Raf, we favor the use of the B‐Raf(V600E), because it is a
variant found in human cancers.
Two types of constitutively activated mutants of Tiam1 have been
described. First, Tiam1 can be activated by N‐terminal truncation of sequences upstream of the catalytic DH domain (designated C1199) (Michiels
et al., 1997). A second type involves a missense mutation (A441G) in the
N‐terminal PH domain. This mutation was identified in human renal cell
cancers, and Tiam1(A441G) was shown to cause transformation of NIH
3T3 cells (Engers et al., 2000).
In addition to laboratory‐generated activated p110‐CAAX, two other
approaches have been identified for activation of this effector pathway that
better mimic mechanisms associated with oncogenesis. One approach
for causing constitutive activation of PI3K involves interfering RNA suppression of PTEN expression. The PTEN tumor suppressor is a lipid
phosphatase that converts the PI3K product phosphatidylinositol 3,4,5‐
trisphosphate (PIP3) to PIP2, and loss of PTEN expression is commonly
seen in human cancers (Steelman et al., 2004). Recently, missense mutation
activated variants of p110 (PIK3CA) have been identified in human
tumors (Samuels et al., 2004) and shown to exhibit transforming activity
(Kang et al., 2005).
Finally, the generation of a C‐terminal Ras plasma membrane–targeted
version of PLC" did show increased lipase activity when overexpressed in
Cos‐7 cells, but unexpectedly this increased activity was not dependent on
the prenylation modification (G. Kelley, unpublished observation).
Constitutively Activated Effector Substrates
Constitutively activated substrates of Ras effectors have also been used
to activate a single effector pathway (Table I). GTPase‐deficient mutants
of Ral and Rac small GTPases, with missense mutations analogous to the
activating mutations found in tumor‐associated Ras proteins (G12V or
Q61L), have been used widely to mimic constitutive activation of Ral
GEFs and Tiam1, respectively. Another type of activated GTPase includes
those with missense mutations that enhance their intrinsic nucleotide
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regulators and effectors of small GTPases: Ras family
[17]
exchange rate (fast cycling mutants) analogous to the F28L fast cycling
mutant of Ras (Reinstein et al., 1991).
Constitutively activated MEKs have been developed that are N‐terminally
truncated and possess missense mutations that mimic phosphorylation by
Raf (Mansour et al., 1994). Weakly activated variants of ERKs have also
been described (Bott et al., 1994; Chu et al., 1996). Although PI3K production of PIP3 can lead to the concurrent activation of many signaling proteins,
constitutively activated mutants of the AKT1 serine/threonine kinase can
mimic the biological consequences of PI3K activation in many situations.
Because PIP3 production promotes AKT1 association with the plasma
membrane where additional phosphorylation events occur to promote full
AKT1 activation, laboratory‐generated plasma membrane–targeted versions of AKT1 (e.g., Myr‐AKT) (Kohn et al., 1996) have been found to act
as activated variants of AKT1.
One caution with the use of activated effectors is that, because effectors can activate multiple substrates, expression of a single activated
substrate may not fully mimic the activity of the activated effector. For
example, RalGEFs activate both RalA and RalB, and, additionally, may
have functions distinct from their activation of these two small GTPases.
Because there is growing evidence that RalA and RalB possess distinct
cellular functions (Chien and White, 2003; Lim et al., 2005; Shipitsin and
Feig, 2004) despite sharing 90% amino acid identity, the cellular consequences of expressing GTPase‐deficient mutants of RalA or RalB may not
result in the same consequences as expression of an activated RalGEF.
Finally, RalGEF activation of Ral may be better mimicked by a fast‐
cycling mutant that is analogous to the fast‐cycling mutants described for
Ras and Rho GTPases (Lin et al., 1997). Similar issues also apply when
using activated substrates of other Ras effectors, and these issues need to
be considered when interpreting the results of experiments using these
reagents.
Reagents for Assessment of Effector Necessity
With the multiplicity of potential effector pathways downstream of Ras
signaling, ascribing a cellular phenotype to a particular effector cascade
requires additional tools. It is imprudent to assume that when constitutive
activation of a pathway leads to a cellular change, that that effector is
necessary. Indeed, because an outcome such as morphological transformation can come about by means of several different (and often synergistic)
pathways, it is necessary to inhibit each individually to reveal which effectors are required as opposed to simply sufficient. Various approaches to
[17] analyses of Ras effector utilization in cellular transformation
203
block the activity of a specific effector pathway have been developed and
are summarized in Table II. In addition to these cell culture–based approaches, recent studies have used mice deficient in the expression of a
particular effector not essential for development (e.g., Tiam1, PLC",
RalGDS) to demonstrate the necessary role of specific effector function
for H‐Ras–mediated skin tumor formation (Bai et al., 2004; Gonzalez‐
Garcia et al., 2005; Malliri et al., 2002).
Pharmacological Inhibitors
Perhaps the most useful and widely used reagents for evaluating
effector‐signaling necessity have been pharmacological inhibitors of MEK
activation of ERK (U0126 and PD98059) (Alessi et al., 1995; Duncia et al.,
1998) and PI3K (LY294002 and wortmannin) (Carpenter and Cantley,
1996). In addition to MEK inhibitors, several inhibitors of Raf have recently been described. First, BAY 43–9006 is an inhibitor of Raf kinase activity,
although potent inhibition of other kinases has also been described
(Wilhelm et al., 2004). In particular, potent inhibition of vascular endothelial growth factor receptors (VEGFR‐2, VEGFR‐3) is seen; thus, this
inhibitor is also described as an angiogenesis inhibitor. With a concentration of 10 M, we have found that BAY 43–9006 blocks cell migration,
invasion through Matrigel (BD Biosciences, Franklin Lakes, NJ), reconstituted basement membrane, and soft agar colony formation of Ras‐
transformed human pancreatic epithelial cells (Campbell, Ouellette, and
Der, unpublished).
MCP compounds were identified and characterized as inhibitors of
Ras interaction with c‐Raf‐1 and were shown to block activated H‐Ras–,
but not Raf‐22W–mediated transformation of NIH 3T3 cells (Kato‐
Stankiewicz et al., 2002). We have used MCP1 and MCP110 (dissolved in
DMSO) at a concentration range of 10–20 M in cell culture experiments
to inhibit the cascade downstream of Raf kinase activation. However,
because the precise mechanism by which MCP compounds block
Ras activation of Raf is currently unresolved, whether their ability to
block Ras transformation of NIH 3T3 cells is due simply to blocking Ras‐
driven activation of Raf is unclear. Finally, a cell‐permeable inhibitor of
RacGEF activation of Rac, NSC23766, has recently been identified (Gao
et al., 2004).
Although inhibitors of protein prenylation (e.g., farnesyltransferase
inhibitors) can be used to block small GTPase function, because they target
enzymes with multiple substrates, they are not very specific inhibitors of
GTPase function (Sebti and Der, 2003).
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[17]
TABLE II
REAGENTS FOR INHIBITION OF RAS EFFECTOR PATHWAYS
Inhibitor
Description
Reference
Raf‐MEK‐ERK pathway
Raf‐301
MEK1(K97A)
MEK2(K101A)
ERK1/p44 (K71R)
ERK2/p42 (K52R)
BAY 43‐9006
MCP110
U0126
PD98059
K375W missense mutation of the ATP
binding, kinase‐deficient mutant
K97A missense mutation of the ATP
binding, kinase‐deficient mutant
K101A missense mutation of the ATP
binding, kinase‐deficient mutant
K71R missense mutation of the ATP
binding site, kinase‐deficient mutant
ATP binding site, kinase‐deficient mutant
Cell‐permeable inhibitor of Raf kinase
activity; also potent inhibition of a
variety of other protein kinases
Cell‐permeable inhibitor of Ras
interaction with c‐Raf‐1 and activation
of ERK
Cell‐permeable inhibitor of MEK
activation of ERK
Cell‐permeable inhibitor of MEK
activation of ERK
(Kolch et al., 1991)
(Seger et al., 1994)
(Abbott and Holt,
1999)
(Robbins et al., 1993)
(Robbins et al., 1993)
(Lyons et al., 2001)
(Kato‐Stankiewicz
et al., 2002)
(Davies et al., 2000)
(Davies et al., 2000)
PI3K‐AKT pathway
Wortmannin
LY294002
PTEN
Cell‐permeable inhibitor of PI3K family
lipid kinases
Cell‐permeable inhibitor of PI3K family
lipid kinases
Lipid phosphatase, converts PIP3 to PIP2
(Davies et al., 2000)
(Davies et al., 2000)
RalGEF‐Ral pathway
RalA(S28N or
S31N)a
RalA(G26A or
G29N)a
RalB(S28N)
RalB(G26A)
RalA siRNA
RalB siRNA
Dominant negative; inhibitor of
activation of Ral
Dominant negative; inhibitor of
activation of Ral
Dominant negative; inhibitor of
activation of Ral
Dominant negative; inhibitor of
activation of Ral
pSUPER.retro.puro retrovirus
expression vector
pSUPER.retro.puro retrovirus
expression vector
RalGEF
(Urano et al., 1996)
RalGEF
(Jullien‐Flores
et al., 1995)
(Urano et al., 1996)
RalGEF
RalGEF
(Jullien‐Flores
et al., 1995)
(Lim et al., 2005)
(Lim et al., 2005)
Tiam1‐Rac pathway
Tiam1 C1199
Rac1(G12V)
N‐terminally truncated, constitutively
activated
GTPase‐deficient mutant
(Michiels et al., 1997)
(Ridley et al., 1992)
[17] analyses of Ras effector utilization in cellular transformation
205
TABLE II (continued)
Inhibitor
Rac1(Q61L)
Description
Reference
GTPase‐deficient mutant
(Xu et al., 1994)
pSUPER.retro retrovirus expression
vector
Unpublished,
G. Kelley, SUNY
Upstate Medical
University
PLC"
PLC" siRNA
a
Two human RalA sequences have been identified, with one containing three additional
N‐terminal amino acids (Chardin and Tavitian, 1989; Polakis et al., 1989).
Dominant Negative Mutants
Another method to illuminate contributing roles of particular pathways
is the use of dominant negative isoforms of GTPases analogous to the
commonly used H‐Ras(S17N) or more potent H‐Ras(G15A) (Chen et al.,
1994) that forms a nonactivating complex with RasGEFs and prevents their
activation of Ras (Feig, 1999). For example, use of a dominant‐negative
RalA implicated the RalGEF effector cascade in Ras transformation of
HEK human embryonic kidney epithelial cells (Hamad et al., 2002). These
dominant negatives block GEF activation of a GTPase but will not block the
activity of GTPase‐deficient mutants of GTPases, which are activated independent of GEF function. A caution regarding these dominant negatives is
that they, in principle, block all GEFs for a particular GTPase. Hence, the
Rac1(S17N) may block the activities of Tiam1, as well as Vav and other
RacGEFs. Therefore, nonspecific activities may be seen with these reagents.
We have also used kinase‐deficient, dominant negative mutants of
c‐Raf‐1, MEK, and ERK to show that the ERK‐MAPK cascade is important for Ras transformation. For example, coexpression of kinase‐deficient
mutants of MEK2 (K101A), ERK1/p44 (K71R) or ERK2/p42(K52R), or
c‐Raf‐1 (K375W) blocked Ras transforming activity (Brtva et al., 1995;
Gupta et al., 2000; Khosravi‐Far et al., 1995). However, their precise mechanism of action and their possible nonspecific effectors make them less
attractive than the use of the pharmacological inhibitors described previously. Similarly, kinase‐deficient mutants of AKT (Kohn et al., 1996), as
well as ectopic expression of the PTEN lipid phosphatase, have been used
to block PI3K activity (Downward, 2004).
Interfering RNA (RNAi)
Recently, the use of interfering RNA has provided a powerful approach to evaluate the contribution of effector signaling components in
206
regulators and effectors of small GTPases: Ras family
[17]
Ras transformation. One limitation of this approach is when there exist
multiple, functionally overlapping isoforms of a particular effector, for
example, the Raf kinases. Suppression of one Raf isoform alone is not
likely to be sufficient to block Ras activation of ERK (Wellbrock et al.,
2004b). However, in situations where there is only one isoform, this approach can be very useful. For example, shown in Fig. 2 are our analyses of
RNAi suppression of PLC" expression in 208F rat fibroblasts. Stable infection of these cells with pSUPER.retro.puro vectors (OligoEngine, Seattle,
WA) expressing short hairpin RNA (shRNA) specific for PLC" significantly reduced endogenous protein expression levels. The sequences used for
the shRNAs for PLC" will be described elsewhere (G. Kelley, in preparation). Surprisingly, we found that Ras transformation was enhanced by the
downregulation of PLC", as measured by colony formation in soft agar
(Singh et al., unpublished).
Interfering RNA (RNAi) has been very useful for the analyses of Ral
GTPase function in Ras transformation (Chien and White, 2003). For
FIG. 2. Use of interfering RNA to evaluate the contribution of PLC" to Ras‐mediated
transformation. (A) Stable suppression of endogenous PLCe in 208F rat fibroblasts infected
with pSUPER.retro retrovirus vectors encoding short hairpin sequences corresponding to
three different sequences of PLC". After infection and selection in puromycin‐supplemented
growth medium, multiple, drug‐resistant colonies were pooled together for Western blot
analyses of endogenous PLC" protein expression using antibody generated against the RA
domains of rat PLC" (Kelley et al., 2001). (B) Enhanced colony formation of Ras‐transformed
208F cells with reduced endogenous PLC" expression.
[17] analyses of Ras effector utilization in cellular transformation
207
example, we recently applied pSUPER.retro.puro retrovirus expression
vectors for stable expression of RNAi specific for human RalA or RalB
(Lim et al., 2005). These analyses showed that RalA was required for
Ras transformation of HEKs, whereas suppression of RalB expression
enhanced Ras transformation, and, furthermore, RalA was important
for the growth of Ras mutation–positive pancreatic and other human
tumor cell lines. These results add further to previous observations that
RalA and RalB possess significant function differences and distinct roles
in oncogenesis.
Retrovirus, Cell Culture, and Effector Expression
Verification Methods
Generation of Infectious Retrovirus
To establish cells stably expressing ectopically introduced genes that
activate a specific effector signaling pathway, we typically use the pBabe
retrovirus vector expression system (Morgenstern and Land, 1990). The
pBabe‐based expression plasmids can be stably introduced into mammalian cells by DNA transfection or, after generation of infectious virus as
described later, by retrovirus infection. We prefer using a retroviral infection system, because the resultant virus has the ability to introduce genes
into human cell types that are often inefficiently transfected. Most activated H‐Ras effector domain mutants, activated effectors, or effector substrates have been subcloned into the pBabe retroviruses (Table II).
Expression of the inserted cDNA sequence is driven by the Moloney
murine leukemia virus (MMuLV) long terminal repeat promoter, and a
second gene encoding for antibiotic resistance (neomycin, hygromycin,
bleomycin, and puromycin) is expressed from the SV40 early promoter.
To generate retrovirus for each pBabe construct, we use the Stratagene
pVPack retrovirus system that can be used with any MMuLV‐based retrovirus vector to produce high titer viral supernatants. We use the highly
transfectable human embryonic kidney epithelial 293T cell line, and we
introduce the three plasmids by calcium phosphate precipitation: the pBabe
expression construct, the CMV‐based pVPack‐GP (encodes viral gag and
pol genes; No. 217566, Stratagene, La Jolla, CA), and either pVPack‐Eco
or pVPack‐Ampho (No. 217569 or No. 217568, respectively, Stratagene)
plasmid DNAs to create infectious but replication‐incompetent viral particles for infection of rodent or human cells, respectively. Information on
these plasmids and general protocols for their use are provided in detail
208
regulators and effectors of small GTPases: Ras family
[17]
from the manufacturer (http://www.stratagene.com/manuals/217566.pdf).
Because virus generated with the amphotropic env protein can infect human
cells, the appropriate safety guidelines need to be followed.
Retroviral Infection
Day 1: 293T cells (maintained in growth medium: Dulbecco’s minimum
essential medium [DMEM] supplemented with 10% fetal calf serum [FCS]
and 1% penicillin/streptomycin) is plated at 106 cells in a T25 flask so that
they are at 60–70% confluency on the second day.
Day 2: Cells are fed with 4 ml of fresh medium containing 25 M
chloroquine 20 min before adding the plasmid DNA mix.
DNA mix: pVPack‐GPol 3, g; pVPack‐Ampho, 3 g; plasmid DNA,
3 g; HBS, 0.9 ml; 0.1 ml of 1.25 M CaCl2 is added, and the mix is incubated
for 10 min at room temperature to allow DNA to precipitate. The DNA
mix is added to s93T cells and incubated at 37 for 3 h. At this point, the
cells are considered to be infectious and must be treated as such. Removal
of medium is now by pipette instead of aspiration to reduce aerosolization
of viral particles. All used pipettes and plastic ware should be bleached
before disposal. Cells are fed with 4 ml fresh growth medium containing 25
M chloroquine and incubated for 6–h and then re‐fed with fresh growth
medium alone for additional overnight incubation at 37 .
Day 3: Target mammalian cells are split into T25 flasks at 20% confluency, and an extra flask is plated for use as a selection control. 293T cells
are re‐fed with 3 ml fresh growth medium.
Day 4: Although the virus‐containing medium from the 293T cells
can be frozen in liquid nitrogen or dry ice and stored at –80 at this point,
it should be noted that the viral titer is significantly reduced by each freeze/
thaw cycle. As a result, it is preferable to coordinate the mammalian
target cells so that they are ready for infection with fresh viral supernatant.
Target cells are fed with 4 ml fresh growth medium containing 8 g/ml
polybrene 20 min before adding virus. Virus‐containing medium is
removed from the 293T cells and filtered through a 0.45‐m low‐protein
binding filter. Target cell growth medium (1.5 ml) and 4 l of 8 g/ml
polybrene are mixed with 2.5 ml of the virus. The old medium is aspirated
from the target cells and replaced with this mix for 3 h at 37 . An extra 2 ml
of target cell growth medium is added, and the cells are incubated
overnight at 37 .
Day 5: The virus‐containing culture supernatant is removed from the
target cells, and they are fed with fresh growth medium.
Days 6 and 7: Cells are selected with 1 g/ml puromycin (or other
selection agent, as required by the retroviral vector). Multiple drug‐resistant
[17] analyses of Ras effector utilization in cellular transformation
209
colonies (>100 cells) are then trypsinized and pooled together to establish
mass populations of stably infected cells.
Verification of Effector Expression and Activation
To analyze the effectiveness and specificity of constitutive Ras expression and effector activation, whole cell lysates are separated by SDS‐
polyacrylamide gel electrophoresis (SDS‐PAGE). Target cells stably
expressing pBabe‐puro expression constructs are plated at a density of
3 105 per 10‐cm dish 24 h before starvation. Cells are washed once with
1 phosphate‐buffered saline (PBS) and grown for 48 h in starvation
medium consisting of DMEM supplemented with 0.5% heat‐inactivated
FCS. Cells are lysed in buffer containing 50 mM Tris (pH 7.5), 150 mM
NaCl, 50 mM NaF, and 1% NP40. Lysates are clarified of membrane
debris by centrifugation at 14,000g for 10 min at 4 before use. Protein
concentrations from total cell lysates are determined using the BCA Protein Assay Kit (No. 23225, Pierce Chemical Co., Rockford, IL), and 20–30
g of total cell lysate is separated by SDS‐PAGE in 10% acrylamide gels
and transferred to Immobilon‐P (No. IPVH00010, Millipore, Bedford,
MA) polyvinyldiflouride membranes. Membranes are then blocked and
incubated in primary antibodies as per the manufacturer. Horseradish
peroxidase–conjugated secondary antibodies (No. NA9310 or NA9314 for
mouse and rabbit, respectively, Amersham Pharmacia Biotech, Uppsala,
Sweden) allow detection by enhanced chemiluminescence (Amersham
Pharmacia Biotech).
Primary antibodies used for Western blot detection of effector activation include those for phosphorylated and activated ERK1/p44 and ERK2/
p42 (E10; Santa Cruz Biotechnology, Santa Cruz, CA) and phosphorylated
and activated AKT (phospho‐AKT Ser473; Cell Signaling Technology,
Beverly, MA). Parallel blots are done with antibodies to total ERK1 and
ERK2 (C‐16; Santa Cruz Biotechnology), and AKT (No. 9272; Cell Signaling, Beverly, MA) to verify equivalent total ERK and AKT expression.
Blot analysis for ‐actin expression is used as a loading control (Sigma
Chemical Co., St. Louis, MO).
Pull‐down analyses are used to determine activation of RalA (example
given following) (Lim et al., 2005), Ras, and Rac small GTPases (Taylor
and Shalloway, 1996; Wolthuis et al., 1998). Expression of an effector
binding domain specific for the GTP‐bound form of the small GTPase in
question, from PAK (PAK‐RBD) and RalBP1 (RalBD), respectively for
Rac and Ral activation analyses) is grown in bacteria and bound to agarose
210
regulators and effectors of small GTPases: Ras family
[17]
beads by glutathione S‐transferase (GST)–glutathione interaction. Lysates
from the cells of choice are incubated with these beads to bind GTP‐loaded
protein. The beads are washed and resuspended in Laemmli sample buffer
before proteins are resolved by SDS‐PAGE.
Ral‐GTP Pull‐Down Assay
Day 1: Mammalian cells for testing Ral‐GTP levels are plated in
complete growth medium. Appropriate negative and positive controls for
this analysis include control NIH 3T3 murine cells and NIH 3T3 cells stably
expressing constitutively activated Rlf‐CAAX, 10 min stimulation with
insulin (Murphy et al., 2002), or human tumor cell lines that have low
(e.g., Colo 587, CFPac‐1) or high (e.g., Capan‐1, T3M4) (Lim et al., 2005)
expression of RalA.
Day 2: A culture (50 ml LB‐amp) of E. coli transformed with the pGEX‐
KG‐RalBD plasmid (a generous gift of Doug Andres) (Shao and Andres,
2000) is grown overnight at 37 with shaking. Glutathione‐sepharose
4B beads (Amersham Biosciences, Piscataway, NJ) are washed with cold
PBS twice, suspended in PBS as a 50% v/v slurry, and stored at 4 . The
target cell growth medium is replaced with low serum (e.g., 0.5% calf
serum)–supplemented medium for 24 h before analyses to reduce the basal
level of serum‐stimulated Ral activation.
Day 3: The overnight culture is diluted into 500 ml LB‐amp and grown
at 37 for 2 h. This culture is dosed with 0.1 mM IPTG (isopropyl‐beta‐D‐
thiogalactopyranoside) for 1.5–2 h at 37 to induce expression of the GST‐
RalBD fusion protein. Bacterial cells are collected by centrifugation of
2000g for 15 min at 4 . The supernatant is removed and the cell pellet
resuspended in 10 ml ice‐cold TNE (100 mM NaCl þ 1 mM phenylmethylsulfonylfluoride in TE). The cell suspension is sonicated (3 10 sec) on ice,
and Triton X‐100 is added to a 1% final concentration. Bacterial membranes are pelleted at 10,000g, and 500 l of washed beads is added to the
cleared lysate. The beads are rocked at RT for 5–10 min at room temperature or 4 for 1 h. The beads are centrifuged and washed 3 with ice‐cold
PBS, and then 1 with NP‐40 lysis buffer (25 mM HEPES, pH 7.5, 150 mM
NaCl, 1% NP‐40, 0.25% Na deoxycholate, 10% glycerol, 10 mM MgCl2
50 l/ml 0.5 M NaF, 2 l/ml 0.5 M EDTA, 10 l/ml NaVO4, 4.55 l/ml
2.2 g/ml aprotinin, 1 l/ml leupeptin). The beads should be stored as a
50% slurry for no more than 2 days before using. To probe the target cell
for RalA GTP loading, 100 g of mammalian cell lysate is rocked with 2–10
g of prepared beads for 30 min at 4 , then spun beads are washed 2 with
NP‐40 lysis buffer, then 1 NP‐40 lysis buffer þ 0.5 M NaCl. Beads are
resuspended in sample buffer, and the eluted protein run on SDS‐PAGE is
transferred to PVDF and blotted for RalA protein (No. 610221; BD
[17] analyses of Ras effector utilization in cellular transformation
211
Transduction Labs, San Diego, CA). Parallel lanes containing equal
amounts of total cell lysate are run to blot for total RalA protein to verify
that the differences seen in RalA‐GTP levels are not due to differences in
total RalA protein expression.
Analysis of Effector Function in Ras‐Mediated Morphological and
Growth Transformation of ROSE Ovarian Epithelial Cells
Although it is well established that oncogenic forms of Ras can promote
cellular transformation and other phenotypic changes implicated in cancer,
not all cell types respond in a similar fashion. In addition, the different
signaling pathways downstream of Ras drive separate cellular effects, often
in a very cell context–dependent manner. The agents described previously
FIG. 3. Raf activation is sufficient to promote Ras‐mediated morphological and growth
transformation of ROSE199 cells. (A) Verification of Ras and activated effector signaling
activity in ROSE199 cells. (B) Constitutive activation of Raf kinase, but not PI3K or RalGEF,
is able to recapitulate at least some of the morphological and contact‐independent growth
changes seen in H‐Ras(12V) cells.
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regulators and effectors of small GTPases: Ras family
[17]
can be used to link a characteristic to a particular effector stream and
concurrently rule out others. For example, although stable expression of
pBabe‐puro H‐Ras(12V) initiates contact‐independent growth in rat ovarian surface epithelial (ROSE) 199 cells, only one of the effector pathways
alone drives soft agar colony formation. Growth in agar was evident for
Raf‐CAAX–transformed ROSE cells (albeit to a lesser degree than H‐Ras
(12V)–transformed cells) but not cells with activated PI3K (p110‐CAAX)
or Ral (Rlf‐CAAX) (Ülkü et al., 2003) (Fig. 3). Similarly, the morphological transformation observed in H‐Ras(12V)–expressing cells was also
seen with Raf‐CAAX but not p110‐CAAX– or Rlf‐CAAX–expressing
ROSE cells, indicating that activation of Raf kinase is sufficient for
this transformed phenotype in these cells. The requirement of Raf‐MEK‐
ERK signaling is demonstrated by using the U0126 inhibitor to block
the ability of activated MEK1 and MEK2, the only currently known substrates of Raf, to phosphorylate ERK1/2. U0126‐treated ROSE199 cells
expressing H‐Ras(12V) failed to grow in soft agar, indicating that the Raf‐
MEK‐ERK axis is necessary for anchorage‐independent growth of these
ovarian cells (Fig. 4). It must be noted that sufficient does not imply
exclusivity, because in the preceding example, although Raf activation
alone does drive soft agar colony formation, it is only approximately 30%
of that seen with H‐Ras(12V), indicating that there are other effector
pathways of Ras signaling contributing to the extent of phenotypical
change.
FIG. 4. The Raf‐MEK‐ERK and PI3K‐AKT pathways are necessary for Ras‐mediated
anchorage‐independent growth.
[17] analyses of Ras effector utilization in cellular transformation
213
Conclusion
As the field of Ras family small GTPase signaling grows, it has become
evident that the pathways are not simply linear cascades from GTPase
through a single effector to target molecule. Consequently, individual
cellular phenotypes cannot be ascribed to only one pathway or effector
molecule because of redundancy, synergism, and cross‐talk. As such, more
discrete and specific molecular tools are needed to tease out the respective
contributions of these many GTPase signaling nodes. In this chapter, we
have described several such tools, both pharmacological and genetic, in use
in our laboratory and others that can help to explain the involvement of
specific facets of the complex Ras protein signaling network in oncogenic
Ras function.
References
Abbott, D. W., and Holt, J. T. (1999). Mitogen‐activated protein kinase kinase 2 activation is
essential for progression through the G2/M checkpoint arrest in cells exposed to ionizing
radiation. J. Biol. Chem. 274, 2732–2742.
Alessi, D. R., Cuenda, A., Cohen, P., Dudley, D. T., and Saltiel, A. R. (1995). PD 098059 is a
specific inhibitor of the activation of mitogen‐activated protein kinase in vitro and in vivo.
J. Biol. Chem. 270, 27489–27494.
Bai, Y., Edamatsu, H., Maeda, S., Saito, H., Suzuki, N., Satoh, T., and Kataoka, T. (2004).
Crucial role of phospholipase Cepsilon in chemical carcinogen‐induced skin tumor
development. Cancer Res. 64, 8808–8810.
Bott, C. M., Thorneycroft, S. G., and Marshall, C. J. (1994). The sevenmaker gain‐of‐function
mutation in p42 MAP kinase leads to enhanced signalling and reduced sensitivity to dual
specificity phosphatase action. FEBS Lett. 352, 201–205.
Brtva, T. R., Drugan, J. K., Ghosh, S., Terrell, R. S., Campbell‐Burk, S., Bell, R. M., and Der,
C. J. (1995). Two distinct Raf domains mediate interaction with Ras. J. Biol. Chem. 270,
9809–9812.
Carpenter, C. L., and Cantley, L. C. (1996). Phosphoinositide kinases. Curr. Opin. Cell Biol.
8, 153–158.
Chardin, P., and Tavitian, A. (1989). Coding sequences of human ralA and ralB cDNAs.
Nucleic Acids Res. 17, 4380.
Chen, S. Y., Huff, S. Y., Lai, C. C., Der, C. J., and Powers, S. (1994). Ras‐15A protein shares
highly similar dominant‐negative biological properties with Ras‐17N and forms a stable,
guanine‐nucleotide resistant complex with CDC25 exchange factor. Oncogene 9,
2691–2698.
Chien, Y., and White, M. A. (2003). RAL GTPases are linchpin modulators of human
tumour‐cell proliferation and survival. EMBO Rep. 4, 800–806.
Chu, Y., Solski, P. A., Khosravi‐Far, R., Der, C. J., and Kelly, K. (1996). The mitogen‐
activated protein kinase phosphatases PAC1, MKP‐1, and MKP‐2 have unique substrate
specificities and reduced activity in vivo toward the ERK2 sevenmaker mutation. J. Biol.
Chem. 271, 6497–6501.
Cox, A. D., and Der, C. J. (2002). Ras family signaling: Therapeutic targeting. Cancer Biol.
Ther. 1, 599–606.
214
regulators and effectors of small GTPases: Ras family
[17]
Davies, H., Bignell, G. R., Cox, C., Stephens, P., Edkins, S., Clegg, S., Teague, J., Woffendin,
H., Garnett, M. J., Bottomley, W., Davis, N., Dicks, E., Ewing, R., Floyd, Y., Gray, K.,
Hall, S., Hawes, R., Hughes, J., Kosmidou, V., Menzies, A., Mould, C., Parker, A.,
Stevens, C., Watt, S., Hooper, S., Wilson, R., Jayatilake, H., Gusterson, B. A., Cooper, C.,
Shipley, J., Hargrave, D., Pritchard‐Jones, K., Maitland, N., Chenevix‐Trench, G., Riggins,
G. J., Bigner, D. D., Palmieri, G., Cossu, A., Flanagan, A., Nicholson, A., Ho, J. W.,
Leung, S. Y., Yuen, S. T., Weber, B. L., Seigler, H. F., Darrow, T. L., Paterson, H., Marais,
R., Marshall, C. J., Wooster, R., Stratton, M. R., and Futreal, P. A. (2002). Mutations of
the BRAF gene in human cancer. Nature 417, 949–954.
Davies, S. P., Reddy, H., Caivano, M., and Cohen, P. (2000). Specificity and mechanism of
action of some commonly used protein kinase inhibitors. Biochem. J. 351, 95–105.
Downward, J. (2004). PI 3‐kinase, Akt and cell survival. Semin. Cell Dev. Biol. 15, 177–182.
Duncia, J. V., Santella, J. B., 3rd, Higley, C. A., Pitts, W. J., Wityak, J., Frietze, W. E., Rankin,
F. W., Sun, J. H., Earl, R. A., Tabaka, A. C., Teleha, C. A., Blom, K. F., Favata, M. F.,
Manos, E. J., Daulerio, A. J., Stradley, D. A., Horiuchi, K., Copeland, R. A., Scherle,
P. A., Trzaskos, J. M., Magolda, R. L., Trainor, G. L., Wexler, R. R., Hobbs, F. W., and
Olson, R. E. (1998). MEK inhibitors: The chemistry and biological activity of U0126, its
analogs, and cyclization products. Bioorg. Med. Chem. Lett. 8, 2839–2844.
Engers, R., Zwaka, T. P., Gohr, L., Weber, A., Gerharz, C. D., and Gabbert, H. E. (2000).
Tiam1 mutations in human renal‐cell carcinomas. Int. J. Cancer 88, 369–376.
Eves, E. M., Xiong, W., Bellacosa, A., Kennedy, S. G., Tsichlis, P. N., Rosner, M. R., and Hay, N.
(1998). Akt, a target of phosphatidylinositol 3‐kinase, inhibits apoptosis in a differentiating
neuronal cell line. Mol. Cell. Biol. 18, 2143–2152.
Fabian, J. R., Daar, I. O., and Morrison, D. K. (1993). Critical tyrosine residues regulate the
enzymatic and biological activity of Raf‐1 kinase. Mol. Cell. Biol. 13, 7170–7179.
Feig, L. A. (1999). Tools of the trade: Use of dominant‐inhibitory mutants of Ras‐family
GTPases. Nat. Cell Biol. 1, E25–E27.
Feig, L. A., and Buchsbaum, R. J. (2002). Cell signaling: Life or death decisions of ras
proteins. Curr. Biol. 12, R259–R261.
Gao, Y., Dickerson, J. B., Guo, F., Zheng, J., and Zheng, Y. (2004). Rational design and
characterization of a Rac GTPase‐specific small molecule inhibitor. Proc. Natl. Acad. Sci.
USA 101, 7618–7623.
Gonzalez‐Garcia, A., Pritchard, C. A., Paterson, H. F., Mavria, G., Stamp, G., and Marshall,
C. J. (2005). RalGDS is required for tumor formation in a model of skin carcinogenesis.
Cancer Cell 7, 219–226.
Gupta, S., Plattner, R., Der, C. J., and Stanbridge, E. J. (2000). Dissection of Ras‐dependent
signaling pathways controlling aggressive tumor growth of human fibrosarcoma cells:
Evidence for a potential novel pathway. Mol. Cell. Biol. 20, 9294–9306.
Hamad, N. M., Elconin, J. H., Karnoub, A. E., Bai, W., Rich, J. N., Abraham, R. T., Der, C. J.,
and Counter, C. M. (2002). Distinct requirements for Ras oncogenesis in human versus
mouse cells. Genes Dev. 16, 2045–2057.
Hancock, J. F. (2003). Ras proteins: Different signals from different locations. Nat. Rev. Mol.
Cell. Biol. 4, 373–384.
Joneson, T., White, M. A., Wigler, M. H., and Bar‐Sagi, D. (1996). Stimulation of membrane
ruffling and MAP kinase activation by distinct effectors of RAS. Science 271, 810–812.
Jullien‐Flores, V., Dorseuil, O., Romero, F., Letourneur, F., Saragosti, S., Berger, R.,
Tavitian, A., Gacon, G., and Camonis, J. H. (1995). Bridging Ral GTPase to Rho
pathways. RLIP76, a Ral effector with CDC42/Rac GTPase‐activating protein activity.
J. Biol. Chem. 270, 22473–22477.
[17] analyses of Ras effector utilization in cellular transformation
215
Kang, S., Bader, A. G., and Vogt, P. K. (2005). Phosphatidylinositol 3‐kinase mutations
identified in human cancer are oncogenic. Proc. Natl. Acad. Sci. USA 102, 802–807.
Kato‐Stankiewicz, J., Hakimi, I., Zhi, G., Zhang, J., Serebriiskii, I., Guo, L., Edamatsu, H.,
Koide, H., Menon, S., Eckl, R., Sakamuri, S., Lu, Y., Chen, Q. Z., Agarwal, S., Baumbach,
W. R., Golemis, E. A., Tamanoi, F., and Khazak, V. (2002). Inhibitors of Ras/Raf‐1
interaction identified by two‐hybrid screening revert Ras‐dependent transformation
phenotypes in human cancer cells. Proc. Natl. Acad. Sci. USA 99, 14398–14403.
Kelley, G. G., Reks, S. E., Ondrako, J. M., and Smrcka, A. V. (2001). Phospholipase C
(epsilon): A novel Ras effector. EMBO J. 20, 743–754.
Khosravi‐Far, R., Solski, P. A., Clark, G. J., Kinch, M. S., and Der, C. J. (1995). Activation of
Rac1, RhoA, and mitogen‐activated protein kinases is required for Ras transformation.
Mol. Cell. Biol. 15, 6443–6453.
Khosravi‐Far, R., White, M. A., Westwick, J. K., Solski, P. A., Chrzanowska‐Wodnicka, M.,
Van Aelst, L., Wigler, M. H., and Der, C. J. (1996). Oncogenic Ras activation of Raf/
mitogen‐activated protein kinase‐independent pathways is sufficient to cause tumorigenic
transformation. Mol. Cell. Biol. 16, 3923–3933.
Kohn, A. D., Takeuchi, F., and Roth, R. A. (1996). Akt, a pleckstrin homology domain
containing kinase, is activated primarily by phosphorylation. J. Biol. Chem. 271,
21920–21926.
Kolch, W., Heidecker, G., Lloyd, P., and Rapp, U. R. (1991). Raf‐1 protein kinase is required
for growth of induced NIH/3T3 cells. Nature 349, 426–428.
Kumar, R., Angelini, S., Snellman, E., and Hemminki, K. (2004). BRAF mutations are
common somatic events in melanocytic nevi. J. Invest. Dermatol. 122, 342–348.
Leevers, S. J., Paterson, H. F., and Marshall, C. J. (1994). Requirement for Ras in
Raf activation is overcome by targeting Raf to the plasma membrane. Nature 369,
411–414.
Lim, K. H., Baines, A. T., Fiordalisi, J. J., Shipitsin, M., Feig, L. A., Cox, A. D., Der, C. J., and
Counter, C. M. (2005). Activation of RalA is critical for Ras‐induced tumorigenesis of
human cells. Cancer Cell 7, 533–545.
Lin, R., Bagrodia, S., Cerione, R., and Manor, D. (1997). A novel Cdc42Hs mutant induces
cellular transformation. Curr. Biol. 7, 794–797.
Lyons, J. F., Wilhelm, S., Hibner, B., and Bollag, G. (2001). Discovery of a novel Raf kinase
inhibitor. Endocr. Relat. Cancer 8, 219–225.
Malliri, A., van der Kammen, R. A., Clark, K., van der Valk, M., Michiels, F., and Collard,
J. G. (2002). Mice deficient in the Rac activator Tiam1 are resistant to Ras‐induced skin
tumours. Nature 417, 867–871.
Malumbres, M., and Barbacid, M. (2003). RAS oncogenes: The first 30 years. Nat. Rev. Cancer
3, 459–465.
Mansour, S. J., Matten, W. T., Hermann, A. S., Candia, J. M., Rong, S., Fukasawa, K., Vande
Woude, G. F., and Ahn, N. G. (1994). Transformation of mammalian cells by
constitutively active MAP kinase. Science 265, 966–970.
McFall, A., Ulku, A., Lambert, Q. T., Kusa, A., Rogers‐Graham, K., and Der, C. J. (2001).
Oncogenic Ras blocks anoikis by activation of a novel effector pathway independent of
phosphatidylinositol 3‐kinase. Mol. Cell. Biol. 21, 5488–5499.
Michiels, F., Stam, J. C., Hordijk, P. L., van der Kammen, R. A., Ruuls‐Van Stalle, L.,
Feltkamp, C. A., and Collard, J. G. (1997). Regulated membrane localization of
Tiam1, mediated by the NH2‐terminal pleckstrin homology domain, is required for
Rac‐dependent membrane ruffling and C‐Jun NH2‐terminal kinase activation. J. Cell Biol.
137, 387–398.
216
regulators and effectors of small GTPases: Ras family
[17]
Morgenstern, J. P., and Land, H. (1990). Advanced mammalian gene transfer: High titre
retroviral vectors with multiple drug selection markers and a complementary helper‐free
packaging cell line. Nucleic Acids Res. 18, 3587–3596.
Murphy, G. A., Graham, S. M., Morita, S., Reks, S. E., Rogers‐Graham, K., Vojtek, A.,
Kelley, G. G., and Der, C. J. (2002). Involvement of phosphatidylinositol 3‐kinase,
but not RalGDS, in TC21/R‐Ras2‐mediated transformation. J. Biol. Chem. 277,
9966–9975.
Polakis, P. G., Weber, R. F., Nevins, B., Didsbury, J. R., Evans, T., and Snyderman, R. (1989).
Identification of the ral and rac1 gene products, low molecular mass GTP‐binding proteins
from human platelets. J. Biol. Chem. 264, 16383–16389.
Reinstein, J., Schlichting, I., Frech, M., Goody, R. S., and Wittinghofer, A. (1991). p21 with a
phenylalanine 28—leucine mutation reacts normally with the GTPase activating protein
GAP but nevertheless has transforming properties. J. Biol. Chem. 266, 17700–17706.
Repasky, G. A., Chenette, E. J., and Der, C. J. (2004). Renewing the conspiracy theory
debate: Does Raf function alone to mediate Ras oncogenesis? Trends Cell Biol. 14,
639–647.
Ridley, A. J., Paterson, H. F., Johnston, C. L., Diekmann, D., and Hall, A. (1992). The small
GTP‐binding protein rac regulates growth factor‐induced membrane ruffling. Cell 70,
401–410.
Robbins, D. J., Zhen, E., Owaki, H., Vanderbilt, C. A., Ebert, D., Geppert, T. D., and Cobb,
M. H. (1993). Regulation and properties of extracellular signal‐regulated protein kinases 1
and 2 in vitro. J. Biol. Chem. 268, 5097–6106.
Rodriguez‐Viciana, P., Warne, P. H., Dhand, R., Vanhaesebroeck, B., Gout, I., Fry, M. J.,
Waterfield, M. D., and Downward, J. (1994). Phosphatidylinositol‐3‐OH kinase as a direct
target of Ras. Nature 370, 527–532.
Rodriguez‐Viciana, P., Warne, P. H., Khwaja, A., Marte, B. M., Pappin, D., Das, P.,
Waterfield, M. D., Ridley, A., and Downward, J. (1997). Role of phosphoinositide 3‐OH
kinase in cell transformation and control of the actin cytoskeleton by Ras. Cell 89,
457–467.
Rodriguez‐Viciana, P., Warne, P. H., Vanhaesebroeck, B., Waterfield, M. D., and Downward,
J. (1996). Activation of phosphoinositide 3‐kinase by interaction with Ras and by point
mutation. EMBO J. 15, 2442–2451.
Samuels, Y., Wang, Z., Bardelli, A., Silliman, N., Ptak, J., Szabo, S., Yan, H., Gazdar, A.,
Powell, S. M., Riggins, G. J., Willson, J. K., Markowitz, S., Kinzler, K. W., Vogelstein, B.,
and Velculescu, V. E. (2004). High frequency of mutations of the PIK3CA gene in human
cancers. Science 304, 554.
Sebti, S. M., and Der, C. J. (2003). Opinion: Searching for the elusive targets of
farnesyltransferase inhibitors. Nat. Rev. Cancer 3, 945–951.
Seger, R., Seger, D., Reszka, A. A., Munar, E. S., Eldar‐Finkelman, H., Dobrowolska, G.,
Jensen, A. M., Campbell, J. S., Fischer, E. H., and Krebs, E. G. (1994). Overexpression of
mitogen‐activated protein kinase kinase (MAPKK) and its mutants in NIH 3T3 cells.
Evidence that MAPKK involvement in cellular proliferation is regulated by phosphorylation of serine residues in its kinase subdomains VII and VIII. J. Biol. Chem. 269,
25699–25709.
Shao, H., and Andres, D. A. (2000). A novel RalGEF‐like protein, RGL3, as a candidate
effector for rit and Ras. J. Biol. Chem. 275, 26914–26924.
Shipitsin, M., and Feig, L. A. (2004). RalA but not RalB enhances polarized delivery of
membrane proteins to the basolateral surface of epithelial cells. Mol. Cell. Biol. 24,
5746–5756.
[17] analyses of Ras effector utilization in cellular transformation
217
Stanton, V. P., Jr., Nichols, D. W., Laudano, A. P., and Cooper, G. M. (1989). Definition of
the human raf amino‐terminal regulatory region by deletion mutagenesis. Mol. Cell. Biol.
9, 639–647.
Steelman, L. S., Bertrand, F. E., and McCubrey, J. A. (2004). The complexity of PTEN:
Mutation, marker and potential target for therapeutic intervention. Expert Opin. Ther.
Targets 8, 537–550.
Stokoe, D., Macdonald, S. G., Cadwallader, K., Symons, M., and Hancock, J. F. (1994).
Activation of Raf as a result of recruitment to the plasma membrane. Science 264,
1463–1467.
Taylor, S. J., and Shalloway, D. (1996). Cell cycle‐dependent activation of Ras. Curr. Biol. 6,
1621–1627.
Ülkü, A. S., Schafer, R., and Der, C. J. (2003). Essential role of Raf in Ras transformation and
deregulation of matrix metalloproteinase expression in ovarian epithelial cells. Mol.
Cancer Res. 1, 1077–1088.
Urano, T., Emkey, R., and Feig, L. A. (1996). Ral‐GTPases mediate a distinct downstream
signaling pathway from Ras that facilitates cellular transformation. EMBO J. 15, 810–816.
Vos, M. D., Ellis, C. A., Elam, C., Ulku, A. S., Taylor, B. J., and Clark, G. J. (2003). RASSF2
is a novel K‐Ras‐specific effector and potential tumor suppressor. J. Biol. Chem. 278,
28045–28051.
Wang, Y., Waldron, R. T., Dhaka, A., Patel, A., Riley, M. M., Rozengurt, E., and Colicelli, J.
(2002). The RAS effector RIN1 directly competes with RAF and is regulated by 14‐3‐3
proteins. Mol. Cell. Biol. 22, 916–926.
Wellbrock, C., Karasarides, M., and Marais, R. (2004a). The RAF proteins take centre stage.
Nat. Rev. Mol. Cell. Biol. 5, 875–885.
Wellbrock, C., Ogilvie, L., Hedley, D., Karasarides, M., Martin, J., Niculescu‐Duvaz, D.,
Springer, C. J., and Marais, R. (2004b). V599EB‐RAF is an oncogene in melanocytes.
Cancer Res. 64, 2338–2342.
Wennstrom, S., and Downward, J. (1999). Role of phosphoinositide 3‐kinase in activation of
ras and mitogen‐activated protein kinase by epidermal growth factor. Mol. Cell. Biol. 19,
4279–4288.
White, M. A., Nicolette, C., Minden, A., Polverino, A., Van Aelst, L., Karin, M., and Wigler,
M. H. (1995). Multiple Ras functions can contribute to mammalian cell transformation.
Cell 80, 533–541.
Wilhelm, S. M., Carter, C., Tang, L., Wilkie, D., McNabola, A., Rong, H., Chen, C., Zhang, X.,
Vincent, P., McHugh, M., Cao, Y., Shujath, J., Gawlak, S., Eveleigh, D., Rowley, B., Liu, L.,
Adnane, L., Lynch, M., Auclair, D., Taylor, I., Gedrich, R., Voznesensky, A., Riedl, B., Post,
L. E., Bollag, G., and Trail, P. A. (2004). BAY 43–9006 exhibits broad spectrum oral
antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases
involved in tumor progression and angiogenesis. Cancer Res. 64, 7099–8109.
Wolfman, J. C., Palmby, T., Der, C. J., and Wolfman, A. (2002). Cellular N‐Ras promotes cell
survival by downregulation of Jun N‐terminal protein kinase and p38. Mol. Cell. Biol. 22,
1589–1606.
Wolthuis, R. M., de Ruiter, N. D., Cool, R. H., and Bos, J. L. (1997). Stimulation of gene
induction and cell growth by the Ras effector Rlf. EMBO J. 16, 6748–6761.
Wolthuis, R. M., Franke, B., van Triest, M., Bauer, B., Cool, R. H., Camonis, J. H.,
Akkerman, J. W., and Bos, J. L. (1998). Activation of the small GTPase Ral in platelets.
Mol. Cell. Biol. 18, 2486–2491.
Xu, X., Barry, D. C., Settleman, J., Schwartz, M. A., and Bokoch, G. M. (1994). Differing
structural requirements for GTPase‐activating protein responsiveness and NADPH
oxidase activation by Rac. J. Biol. Chem. 269, 23569–23574.